Cell membranes of various vertebrate cells catalyze a Na+ + K+ + 2Cl- cotransport specifically inhibitable by furosemide and other high ceiling diuretics. The energetics of this process is not elucidated unequivocally. It was clearly shown that cotransport is no ATP-consuming process. We assume that transport is secondary active functionally coupled to the operation of the electrogenic Na+-K+ pump. The role of this transport system in transepithelial ion movement is that it serves as flux amplifier, doubling from 6 to 12 the number of osmotically active particles transported per ATP hydrolyzed. In concert with Na+-K+ pump, cotransport provokes net uptake of KCl into the cell and therefore cellular swelling. This process is regulated by a feedback control system for cell volume; if actual volume reaches reference value, cotransport is switched off to prevent further swelling. How cell volume is measured is not known, nor is the nature of the signal generated to switch cotransport from the operating to the nonoperating state or vice versa. cAMP-level or intracellular Ca2+ play no role as signals or as part of the volume-sensoring mechanism. Theophylline, other alkylxanthines, and some purine ribosides influence cotransport indirectly by reducing reference volume. The role of cytoskeleton in volume regulation is obscure. While high Concentrations of cytochalasin B and of colchicin do not influence cell volume, it is reduced by vinblastine and also by lectins, for example concanavalin A. Volume reduction is accompanied by reduction in cellular KCl content. The observation that during hypertonic incubation protein synthesis is inhibited can be traced back to a correlation between cell volume and protein synthesis and not to elevation of osmolarity per se. Reduction in cell volume under isotonic conditions by varying K+ and/or Cl- concentration or by furosemide inhibition of cotransport is strongly correlated to inhibition of protein synthesis. The reason for this correlation is not yet clarified. Not all cells showing furosemide-sensitive cotransport are able to regulate it, for example lymphocytes. For mammalian erythrocytes drastic species differences exist; while cells from man, rabbit, rat, and mouse all show cotransport, only cells from rat (and mouse?) are able to regulate cotransport.

The existence of an electrogenic Naf pump in Ehrlich cells which substantially contributes t o the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP+), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondria1 membrane potential, by adding t o the overall potential as measured in this way is likely t o cause an overestimation of the membrane potential difference (p.d.). But since this error tends t o diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of a n adequate Na+ concentration gradient. Accordingly, the FZ-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP' at constant Na' concentrations, suggesting that each responds directly t o membrane potential. There is evidence that the electrogenic (free) movement of CI-is slow, at least at normal p.d., whereas a major part of the CI-movement across the cellular membrane appears t o occur by an electrically silent CI--base exchange mechanism. By such a mode CI-, together with an almost stoichiometric amount of K+, may under certain conditions move into the cell against a high adverse electrical potential difference. This "paradoxical" movement of KfC1-contributing t o the deviation of the C1-distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide.As a likely explanation a H+-K+ exchange p u m p was previously offered, even though unequivocal evidence of such a pump is so far lacking. According t o available evidence the electrogenic movement of free CI-is t o o small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. T h e evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na+ concentration gradient, the electrogenic Na+ pump will contribute sufficient extra driving force t o actively transport amino acid into the cells.Key words: amino acid transport, gradient hypothesis, electrogenic cation pump, electrolyte movements, ouabain, furosemideThe active uptake of neutral amino acids b y Ehrlich cells, as in other animal cells and tissues, is according t o many investigators secondary active transport, i.e., driven b y the electrochemical potential gradient of Nat via cotransport (gradient hypothesis),

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Suspension cultures of Digitalis lanata strain I were grown in a medium containing 3% mannitol. For cryopreservation cell suspensions were treated with a mixture of sucrose-glycerol (20%/20 V%), cooled slowly (about 1 degrees C/min) till -100 degrees C and then were transferred to liquid nitrogen. After storage in liquid nitrogen the cells were thawed rapidly in a water bath of 40 degrees C and spread on the surface of a solidified nutrient medium. After 7 days of regrowth the cells were suspended in liquid nutrient medium for further cultivation. About 50% of the cells survived freezing and thawing. However, also the apparently surviving cells showed signs of injury (membrane vesicles outside the plasmalemma, dilated ER cisternae and separation of the nuclear membranes). The cultures derived from the surviving cells had the same growth rate and biochemical activity relative to the transformation of cardenolides, e.g., digitoxin, as the parent cultures. The frequency distribution of the nuclear DNA content in the cell cultures was the same before and after cryopreservation. These results indicate that there is no selection of a special cell type during freezing and thawing.